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Cu2+胁迫对罗氏沼虾血细胞毒性及caspase-3、α-2M基因表达的影响
Effects of Cu2+ Stress on Hemocytes Toxicity and caspase-3, α-2M Gene Expression in Macrobrachium rosenbergii
郭慧, 朱晓闻, 欧荣华, 卢芷程, 谭翠婷, 申玉春, 朱春华*
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DOI:10.11984/j.issn.1000-7083.20170035
作者单位:广东海洋大学水产学院, 湛江市海洋生态与养殖环境重点实验室, 广东湛江 524025
中文关键字:罗氏沼虾;Cu2+;血细胞毒性;基因表达;细胞凋亡
英文关键字:Macrobrachium rosenbergii; Cu2+; hemocytes toxicity; gene expression; apoptosis
中文摘要:本研究以Cu2+(0、100 μg·L-1)对罗氏沼虾Macrobrachium rosenbergii进行处理;分别在胁迫后的0 h、3 h、6 h、12 h、24 h和48 h收集血淋巴;检测血细胞中的细胞凋亡率、活性氧(ROS)含量、酯酶活性、一氧化氮(NO)含量以及细胞凋亡执行分子caspase-3和免疫因子α-2M基因的表达。结果显示;血细胞凋亡率和ROS含量的变化趋势一致;在胁迫后的6~48 h显著升高(P<0.05)。酯酶活性在胁迫后的3~48 h均显著下降(P<0.05);最低值出现在48 h。在胁迫后的6~48 h;NO含量持续增加;并与对照组的差异有统计学意义(P<0.05)。caspase-3的相对基因表达量在胁迫后的6~48 h均显著升高(P<0.05);并在24 h达到最高值。在胁迫后的12 h;α-2M基因的表达量显著升高(P<0.05)并达到峰值;在24 h有短暂降低;但与对照组相比仍显著增加(P<0.05);在48 h α-2M基因的表达量显著降低(P<0.05)。相关性分析显示;血细胞凋亡率和NO以及ROS都呈显著的正相关(P<0.001)。实验表明;Cu2+胁迫显著抑制了机体的酯酶活性;诱导NO和ROS的产生以及免疫相关基因α-2M的表达来对抗Cu2+胁迫带来的损伤。随着胁迫时间的延长;过高浓度的NO和ROS对机体自身造成了伤害;并诱导凋亡相关基因caspase-3的表达;最终导致细胞凋亡;说明Cu2+对虾类具有免疫抑制性和毒性。NO和ROS的升高可能是细胞凋亡的主要诱因。
英文摘要:The present study analyzed the toxic effects of Cu2+ on apoptotic cell ratio, ROS production, esterase activities, NO production, and the expression levels of caspase-3 and α-2M in hemocytes of Macrobrachium rosenbergii. The results showed that the changes of apoptotic cell ratio were consistent with ROS production which was increased significantly (P<0.05) after exposure for 6~48 h. Esterase activities were significantly decreased (P<0.05) at 3~48 h and the minimum was observed at 48 h. The NO production continued increasing and the level became significant difference (P<0.05) after exposure for 6~48 h. The relative expression levels of caspase-3 were significantly induced (P<0.05) after exposure for 6~48 h, and reached the peak at 24 h. The relative expression of α-2M was significantly increased (P<0.05) and reached the peak at 12 h. Moreover, the expression level of α-2M reduced after exposure for 24 h and became significantly different(P<0.05) at 48 h. Correlation analysis showed that significant positive correlation (P<0.001) between apoptotic cell ratio and ROS production was observed, as well as apoptotic cell ratio and NO production. All these results suggested that esterase activities were significantly inhibited after Cu2+ exposure, while NO and ROS production as well as α-2M expression were induced against Cu2+ exposure. Along with stress time increasing, overfull production of ROS and NO caused oxidative stress to prawn and then induced the expression level of caspase-3 which finally resulted in apoptosis, indicating that Cu2+ stress may have immunosuppressive and toxic effects to M. rosenbergii, and the increased production of NO and ROS might be the main cause of apoptosis.
2017,36(3): 293-299 收稿日期:2017-02-08
DOI:10.11984/j.issn.1000-7083.20170035
分类号:X503.225;Q959.223
基金项目:国家自然科学基金项目(31600321);广东省自然科学基金项目(2015A030310438);广东海洋大学创新强校工程科研项目(GDOU2016050253);广东海洋大学优秀青年骨干教师特别资助计划项目(HDYQ2015003)
作者简介:郭慧(1986-),女,讲师,博士,主要从事水产动物健康养殖生态学研究,E-mail:guohuivivian@163.com
*通讯作者:朱春华,男,教授,主要从事水域生态与养殖环境研究,E-mail:zhu860025@163.com
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